In this project we are examining the various cellular and molecular processes that occur during the differentiation of IgA B cells, the main type of B cell occurring in the mucosal immune system. In one phase of this study we transfected CH12.LX B cells with plasmid vectors containing dexamethasone-inducible cassettes producing oligonucleotides that were either sense, anti-sense or non-sense to the alpha-GLT transcription start site (Ialpha start site), and then measured the effect of such transfection on IL-4/TGF-beta-induced alpha-GLT levels and IgA switch differentiation. We found that cells transfected with anti-sense oligonucleotide-producing plasmids, but not cells transfected with sense or non-sense oligonucleotide-producing plasmids, exhibited reduced alpha- GLT levels, as well as decreased membrane IgA expression and IgA secretion. Thus, alpha-GLT appears to be necessary for successful IgA switch differentiation. In a second phase of the study, we measured the expression of RAG genes (recombination activation genes) in mucosal and system lymphoid tissues. Using RT-PCR, we found that B cells in murine Peyer's patches, but not in spleen or lymph nodes, express the RAG-2 gene, but not the RAG-1 gene. Spleen cells cultured in IL-4 alone also express RAG-2 mRNA; this, plus the fact that RAG-2 is expressed in Peyer's patch B cells suggests that the RAG-2 gene product is necessary for certain forms of isotype differentiation. Finally, we studied the activation requirements of post-switch sIgA-bearing B cells. In these studies we showed that whereas sIgM/sIgD-bearing B cells proliferate and produce Ig when stimulated with anti-IgD-dextran, both sIgG-bearing B cells from peripheral lymph nodes and sIgA-bearing B cells from Peter's patches respond with neither proliferation nor secretion (of IgG or IgA, respectively) when stimulated with anti-IgG-dextran or anti-IgA- dextran, respectively. In contrast, such cells do proliferate and secrete when stimulated by T cells. These studies show that isotype-switched B cells require a signal from T cells to undergo terminal differentiation.